Cytiva Bioprocess R&d Ab

Ge Healthcare Bioprocess R&d Ab

Ge Healthcare Bio-Sciences Ab

Annika Forss

Tomas Bjorkman

Mats Ander

Jesper Ulf Hansson

Jean-Luc Maloisel

Gustav Jose Rodrigo

Bo-Lennart Johansson

Gunnar Glad

Hans J Johansson

Carina Engstrand

Gustav José Rodrigo

Hans Blom

The invention relates to a method of isolating an immunoglobulin, comprising the steps of: a) providing a separation matrix comprising multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to a porous support: b) contacting a liquid sample comprising an immunoglobulin with the separation matrix; c) washing said separation matrix with a washing liquid; d) eluting the immunoglobulin from the separation matrix with an elution liquid, and e) cleaning the separation matrix with a cleaning liquid, wherein the alkali-stabilized Protein A domains comprise mutants of a parental Fc-binding domain ofProtein A (SpA).

Separation method

The present invention concerns a method of cleaning and/or sanitizing a separation matrix comprising multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to a porous support. The method comprises the steps of: a) optionally purifying a mixture comprising a first immunoglobulin using the separation matrix; b) providing a cleaning liquid comprising at least 50% by volume of an aqueous alkali metal hydroxide solution; and c) cleaning and/or sanitizing the separation matrix by contacting the cleaning liquid with the separation matrix for a predetermined contact time. The alkali-stabilized Protein A domains comprise mutants of a parental Fc-binding domain ofProtein A (SpA), as defined by SEQ ID NO 51 or SEQ ID NO 52, wherein the amino acid residues at positions 13 and 44 of SEQ ID NO 51 or 52 are asparagines and wherein at least the asparagine residue at position 3 of SEQ ID NO 51 or 52 has been mutated to an amino acid selected from the group consisting of glutamic acid, lysine, tyrosine, threonine, phenylalanine, leucine, isoleucine, tryptophan, methionine, valine, alanine, histidine and arginine.

Method of cleaning and/or sanitizing a separation matrix

The present invention concerns a method of storing a separation matrix comprising multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to a porous support. The method comprises the steps of: a) providing a storage liquid comprising at least 50% by volume of an aqueous alkali metal hydroxide solution; b) permeating the separation matrix with the storage liquid; and c) storing the storage liquid-permeated separation matrix for a storage time of at least days. The alkali-stabilized Protein A domains comprise mutants of a parental Fc-binding domain ofProtein A (SpA), as defined by, or having at least 80% such as at least 90%, 95% or 98% identity to, SEQ ID NO 51 or SEQ ID NO 52, wherein the amino acid residues at positions 13 and 44 of SEQ ID NO 51 or 52 are asparagines and wherein at least the asparagine residue at position 3 of SEQ ID NO 51 or 52 has been mutated to an amino acid selected from the group consisting of glutamic acid, lysine, tyrosine, threonine, phenylalanine, leucine, isoleucine, tryptophan, methionine, valine, alanine, histidine and arginine.

Method of storing a separation matrix

The invention relates to a method of isolating an immunoglobulin, comprising the steps of:

The present invention relates to a chromatography ligand defined by the following formula R&#x2014;R&#x2014;N(R)&#x2014;R&#x2014;Rwherein Ris a substituted or non-substituted phenyl group; Ris a hydrocarbon chain comprising 0-4 carbon atoms; Ris a hydrocarbon chain comprising 1-3 carbon atoms; Ris a hydrocarbon chain comprising 1-5 carbon atoms; and Ris OH or H. The invention also comprises a separation matrix, comprising the described ligands coupled to a porous support, such as particles or a membrane. The ligand and matrix according to the invention is useful for purification of biomolecules or organic compounds, such as proteins, polypeptides, DNA etc. An advantageous use according to the invention is the purification of antibodies.

Chromatography ligand

The invention relates to a method of isolating an immunoglobulin, comprising the steps of: a) providing a separation matrix comprising multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to a porous support, b) contacting a liquid sample comprising an immunoglobulin with the separation matrix, c) washing said separation matrix with a washing liquid, d) eluting the immunoglobulin from the separation matrix with an elution liquid, and e) cleaning the separation matrix with a cleaning liquid, wherein the alkali-stabilized Protein A domains comprise mutants of a parental Fc-binding domain ofProtein A (SpA), as defined by, or having at least 80% such as at least 90%, 95% or 98% identity to, SEQ ID NO 51 or SEQ ID NO 52, wherein the amino acid residues at positions 13 and 44 of SEQ ID NO 51 or 52 are asparagines and wherein at least the asparagine residue at position 3 of SEQ ID NO 51 or 52 has been mutated to an amino acid selected from the group consisting of glutamic acid, lysine, tyrosine, threonine, phenylalanine, leucine, isoleucine, tryptophan, methionine, valine, alanine, histidine and arginine.

The present invention relates to a chromatography ligand defined by the following formula (I) R&#x2014;R&#x2014;N(R)&#x2014;R&#x2014;Rwherein Ris a substituted or non-substituted phenyl group; Ris a hydrocarbon chain comprising 0-4 carbon atoms; Ris a hydrocarbon chain comprising 1-3 carbon atoms; Ris a hydrocarbon chain comprising 1-5 carbon atoms; and Ris OH or H. The invention also comprises a separation matrix, comprising the described ligands coupled to a porous support, such as particles or a membrane. The ligand and matrix according to the invention is useful for purification of biomolecules or organic compounds, such as proteins, polypeptides, DNA etc. An advantageous use according to the invention is the purification of antibodies.

The present invention relates to a method of separating antibodies from other compound(s) in a liquid sample, wherein a mobile phase comprising said sample is contacted with a multi-modal separation matrix to adsorb undesired compounds while the antibodies remain free in the liquid, wherein the multi-modal separation matrix comprises first groups, which are capable of interacting with negatively charged sites of the target compounds, and second groups, which are capable of at least one interaction other than charge-charge interaction with said target compounds. The invention also relates to a chromatography column packed with the above-described multi-modal separation matrix and a filter having such multi-modal groups adsorbed to its surface.

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Uppsala, Sweden

Annika Forss